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Oxidative Damage Enzymes
E. coli Endonuclease III
E. coli Endonuclease VIII
Human 8-oxo-Guanine DNA
Glycosylase (hOGG1)
E. coli Fpg Protein
| Endonuclease III releases damaged
bases induced by UV, ionizing radiation, osmium tetroxide, or acid. It
is a DNA glycosylase with an associated AP lyase activity and contains
an iron-sulfur group which helps to maintain its three dimensional
conformation. Endonuclease III cleaves as a DNA lyase at abasic sites by
b-elimination. This results from a b elimination reaction, producing a
single nucleotide gap in the DNA. The enzyme has a molecular weight of
23.4 kDa and is suitable for use in
FLARE™.
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| The amount of enzyme required to cleave 1 pmole
of a 32P-labeled oligonucleotide probe containing an AP site
within an oligonucleotide duplex in one hour at 37°C. |
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| Purified from E. coli
containing a recombinant plasmid harboring the E. coli
nth gene. |
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| 1X REC™
Buffer 4 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, and 10 mM EDTA),
4 pmoles of AP site oligonucleotide (Cat # 3851-100-01) labeled
with 32P, 4 pmoles of Oligo Complement B (Cat #
3849-100-02), and serial dilutions of enzyme in a 20 µl reaction
volume are incubated for 1 hour at 37°C. For analysis, 5 µl of
REC™ 5X Loading Buffer (10 mM
HEPES-KOH (pH 7.4)), 10 mM EDTA, 6 M urea, 50% glycerol, and
0.2% bromophenol blue) are added, and the tubes are heated at
95°C for 5 min then fast-cooled to 4°C. Cleavage products are
resolved by 20% denaturing polyacrylamide gel electrophoresis. |
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| Freeze in working aliquots at -20°C
in a manual defrost freezer to avoid repeated freeze-thawing.
The enzyme is stored in 10 mM HEPES-KOH (pH 7.4), 100 mM KCl,
0.1 mg/ml BSA, and 50% glycerol. |
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Endonuclease III
catalyzes the excision of the following forms of DNA damage:
Cis- and trans-thymine glycol, 5,6-dihydrothymine,
5,6-dihydroxydihydrothymine, urea
5-hydroxy-5-methylhydantoin, methyltartronylurea,
6-hydroxy-5,6-dihydropyrimidines, 5-hydroxycytosine and
5-hydroxyuracil, 5-hydroxy-6-hydrothymine, 5,6-dihydrouracil
alloxan, uracil glycol, 5-hydroxy-6-hydrouracil, AP sites
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Products
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4045-01K-EB |
E. coli
Endonuclease III, 1000 Units and REC™ Buffer 4, 1 ml |
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4045-05K-EB |
E. coli
Endonuclease III, 5000 Units and REC™ Buffer 4, 5 ml |
| Endonuclease VIII is a class I AP
endonuclease with an associated N-glycosylase activity specific for a
number of modified bases including b-ureidoisobutyric acid and DNA
containing urea. Endonuclease VIII contains ß, δ lyase activity. |
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| One unit is the amount of enzyme required to
cleave an AP site oligonucleotide within an oligonucleotide
duplex at the rate of 1 pmol/hour at 37°C. |
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| Purified from
E. coli containing a recombinant plasmid harboring the
E. coli nei gene. |
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| 1 pmole of
32P-AP-oligonucleotide probe, 1 pmole of complementary
oligonucleotide, 1X REC™
Buffer 3 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, and 1 mM EDTA)
for 1 hour at 37°C in a reaction volume of 20 µl. Cleavage
products are resolved by 20% denaturing polyacrylamide gel
electrophoresis. |
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| Freeze in working aliquots at -20°C
in a manual defrost freezer to avoid repeated freeze-thaws. The
enzyme is supplied in buffer containing 50% glycerol. |
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Products
| 4060-01K-EB |
E. coli Endonuclease VIII, 1000 Units and 10X REC™
Buffer 3, 1 ml |
| 4060-05K-EB |
E. coli Endonuclease VIII, 5000 Units and 10X REC™
Buffer 3, 5 ml |
| Spontaneous oxidation of guanine
residues in DNA generates 8-oxoguanine (8-oxoG). By mispairing with
adenine during replication, 8-oxoG gives rise to G/C to T/A
transversions, a frequent somatic mutation in human cancers. The enzyme,
human 8-oxoguanine DNA glycosylase (hOGG1), recognizes 8-oxoG/C, FaPy/C,
and to a lesser extent, 8-oxoG/T base pairs, catalyzing the removal of
the 8-oxoG and cleavage of the DNA phosphodiester bond through Schiff
base chemistry. Mutations in the hOGG1 gene have been reported in a
small but significant number of transformed cells and tumors. One
mutation, R154H, converts hOGG1 to a promutator by broadening the
specificity of the enzyme for the base opposite 8-oxoG. |
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| Purified from E. coli
containing a recombinant plasmid harboring the hOGG1 gene. |
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One unit of
hOGG1 catalyzes the cleavage of 1 pmole of a
32P-oligonucleotide probe in 1 hour at 37°C at an
8-oxoG/C base pair within an oligonucleotide duplex. |
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| 4 pmoles of
32P-8-oxoG oligonucleotide probe, 4 pmoles of
complementary oligonucleotide, 1X REC™
Buffer 6 (20 mM Tris-Cl (pH 8.0), 1 mM DTT, 1 mM EDTA, 0.1 mg/ml
BSA) for 1 hour at 37°C in a reaction volume of 20 µl. Cleavage
products are resolved by 20% denaturing polyacrylamide gel
electrophoresis and detected by autoradiography. |
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| Store in working aliquots at -20°C
in a manual defrost freezer to avoid repeated freeze-thaws. The
enzyme is supplied in 20 mM Tris-Cl (pH7.8), 1 mM EDTA, 100 mM
NaCl, 1 mM DTT, 50% glycerol. |
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Products
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4130-100-EB |
Human
hOGG1 Protein, 100 Units and 10X REC™ Buffer 6, 1 ml |
| Fpg releases damaged bases
preferentially from duplex DNA. It has an associated class I AP lyase
activity, leaving both 3’ and 5’ phosphoryl groups. This results from a
β, δ elimination reaction at the AP sites, producing a single nucleotide
gap in the DNA. The enzyme consists of 269 amino acids with a molecular
weight of 30.2 kDa. |
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| E. coli Fpg is purified
from E. coli containing a recombinant plasmid harboring
the E. coli Fpg+ gene.
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| One unit is the
amount of enzyme required to cleave 1 pmole of a 32P-labeled
oligonucleotide probe containing 8-oxoguanine within an
oligonucleotide duplex in one hour at 37°C. |
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| 4 pmoles of
32P-8-oxo-dG-oligonucleotide probe, 4 pmoles of
complementary oligonucleotide, 1X REC™
Buffer 10 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 10 mM EDTA, and
0.1 mg/ml BSA) for 1 hour at 37°C in a reaction volume of 20 µl. |
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| Freeze in working aliquots at -80°C
to avoid repeated freeze-thawing. Fpg is supplied in 10 mM
HEPES-KOH, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, and 50%
glycerol. |
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| E. coli Fpg catalyzes the
excision of the following forms of DNA damage:
Open ring forms of 7-methylguanine, including
2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine and
4,6-diamino-5-formamidopyrimidine, a lethal lesion;
8-oxoguanine, a highly mutagenic lesion and probably the most
important biological substrate of Fpg; 5-hydroxycytosine;
5-hydroxyuracil; Aflatoxin-bound imidazole-ring-opened guanine;
Imidazole ring opened N-2-aminofluorene-C8-guanine.
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Products
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4040-100-EB |
E. coli
Fpg Protein, 500 Units and 10X REC™ Buffer 10, 1 ml |
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4040-500-EB |
E. coli Fpg Protein, 2500 Units and 10X REC™ Buffer 10, 1 ml |
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